Repression via Facultative Heterochromatin

项目摘要 表观遗传学/哺乳动物基因调控领域的一个关键问题是细胞身份是如何遗传的 在细胞分裂过程中被后代细胞吸收。表观遗传调控的这一基本方面最近在 我们的实验室：被抑制的，但不是活跃的，染色质结构域是遗传的。抑制的染色质结构域在兼性 异染色质由多亚基复合物，多梳抑制复合物2（PRC 2）维持， 催化组蛋白翻译后修饰H3 K27 me 3。PRC 2展示了一个值得注意的“读和写” H3 K27 me 3的识别导致其变构激活。因此，PRC 2可以完全恢复 H3 K27 me 3-核小体遗传后抑制染色质结构域。值得注意的是，我们的发现指出， 先前报道的组蛋白伴侣NPM 1促进了这种被抑制的染色质的遗传：NPM 1是 当被抑制的染色质被复制时，仅定位于S期晚期的染色质，并直接相互作用 在PRC 2。我们最新的研究结果表明，PRC 2调节基因在生长素作用下的特异性去抑制作用， 在细胞周期的S期期间介导NPM 1的消耗。我们将扩大我们的机械研究， 通过研究NPM 1作为S期特异性组蛋白伴侣的作用及其 在体外用不同候选物进行的一系列组蛋白伴侣测定中与PRC 2相互作用 寡核体模板。我们将研究NPM 1在表观遗传中的作用， 作为NPM 1和相关NPM 1突变体存在的函数的染色质结构域遗传的体内测定。 在复制分叉的上下文中，NPM 1和PRC 2的交互动态是实现以下目标的关键信息： 了解亲本核小体向子DNA链的转移。因此，单分子定位 显微镜以及随机光学重建显微镜（STORM）预计将直接影响 NPM 1的作用及其与表观遗传调节因子PRC 2相互作用的意义。重要的是，突变体 与约35%的急性髓细胞性白血病（AML）相关的NPM 1c错误定位于细胞质。我们 NPM 1c阻碍了PRC 2的正常功能。事实上，类似于我们在NPM 1上的发现， 在NPM 1c AML中，PRC 2的已知HOX基因靶点异常表达，参与了NPM 1c AML的基因缺失。 建立白血病状态。通过PRC 2的H3 K27 me 3沉积和通过DNMT 3A的DNA甲基化结果 但通常是相互排斥的。然而，NPM 1和DNMT 3A突变协同作用， 白血病发生因此，我们进一步提出，DNMT 3A部分补偿了我们提出的NPM 1c。 介导的PRC 2突变的阻碍，其在DNMT 3A突变时丢失。通过NPM 1c的时间表达， 突变体DNMT 3A存在的功能，我们将跟踪对基因表达的影响， 抑制染色质结构域，PRC 2染色质占有率和DNA甲基化，在组织培养和 AML小鼠模型，以充分掌握异常表观遗传事件的序列，因为它们发生在白血病。

Project Summary A critical question in the field of Epigenetics/Mammalian Gene Regulation is how a cellular identity is inherited by progeny cells during cell division. This fundamental aspect of epigenetic regulation was recently clarified in our lab: repressed, but not active, chromatin domains are inherited. Repressed chromatin domains in facultative heterochromatin are maintained by the multi-subunit complex, Polycomb Repressive Complex 2 (PRC2), that catalyzes the histone post-translational modification, H3K27me3. PRC2 exhibits a notable “read and write” feature whereby its recognition of H3K27me3 results in its allosteric activation. Thus, PRC2 can fully restore repressive chromatin domains upon inheritance of H3K27me3-nucleosomes. Remarkably, our findings point to a previously reported histone chaperone, NPM1, as facilitating this inheritance of repressed chromatin: NPM1 is exclusively localized to chromatin in late S-phase when repressed chromatin is replicated, and interacts directly with PRC2. Our latest findings demonstrated specific de-repression of PRC2-regulated genes upon auxin- mediated depletion of NPM1 during S-phase of the cell cycle. We will expand our mechanistic studies of epigenetic inheritance by investigating the role of NPM1 as an S-phase-specific histone chaperone and its interplay with PRC2 in a series of histone chaperone assays performed in vitro with distinct candidate oligonucleosomal templates. We will investigate the role of NPM1 in epigenetic inheritance by adapting our in vivo assay for chromatin domain inheritance as a function of the presence of NPM1 and pertinent NPM1 mutants. The interactive dynamics of NPM1 and PRC2 in the context of a replication fork is critical information towards understanding the transfer of parental nucleosomes to daughter DNA strands. Thus, single-molecule localization microscopy as well as stochastic optical reconstruction microscopy (STORM) are expected to bear directly on the role of NPM1 and the significance of its interaction with the epigenetic regulator, PRC2. Importantly, mutant NPM1c associated with ~35% of all Acute Myelogenous Leukemia (AML) is mis-localized to the cytoplasm. We propose that NPM1c hampers normal PRC2 function. Indeed, similar to our findings above upon NPM1 depletion, known HOX gene targets of PRC2 are aberrantly expressed in NPM1c AML, participating in the establishment of the leukemic state. Deposition of H3K27me3 by PRC2 and DNA methylation by DNMT3A result in repressed chromatin, but are usually mutually exclusive. Yet, NPM1 and DNMT3A mutations synergize in leukemogenesis. Thus, we further propose that DNMT3A partially compensates for our proposed NPM1c- mediated thwarting of PRC2⏤which is lost upon DNMT3A mutation. Through temporal expression of NPM1c as a function of the presence of mutant DNMT3A, we will track the repercussions to gene expression, features of repressed chromatin domains, PRC2 chromatin occupancy and DNA methylation in both tissue culture and a mouse model of AML to fully grasp the sequence of aberrant epigenetic events as they occur in leukemogenesis.